WO2001027255A2 - Famille genique pour ctage - Google Patents

Famille genique pour ctage Download PDF

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WO2001027255A2
WO2001027255A2 PCT/DE2000/003628 DE0003628W WO0127255A2 WO 2001027255 A2 WO2001027255 A2 WO 2001027255A2 DE 0003628 W DE0003628 W DE 0003628W WO 0127255 A2 WO0127255 A2 WO 0127255A2
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Prior art keywords
nucleic acid
protein
ctage
tumor
dna
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WO2001027255A3 (fr
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Stefan EICHMÜLLER
Dirk Schadendorf
Dirk Usener
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Deutsches Krebsforschungszentrum DKFZ
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Deutsches Krebsforschungszentrum DKFZ
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Priority to DE50014417T priority Critical patent/DE50014417D1/de
Priority to EP00983011A priority patent/EP1230265B1/fr
Priority to AU19936/01A priority patent/AU1993601A/en
Priority to US10/110,807 priority patent/US7314922B1/en
Publication of WO2001027255A2 publication Critical patent/WO2001027255A2/fr
Publication of WO2001027255A3 publication Critical patent/WO2001027255A3/fr
Anticipated expiration legal-status Critical
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4748—Tumour specific antigens; Tumour rejection antigen precursors [TRAP], e.g. MAGE
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/53—DNA (RNA) vaccination
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies

Definitions

  • the present invention relates to a new gene family for "cutaneous T-cell lymphoma associated genes" (CTAGE).
  • CTAGE cutaneous T-cell lymphoma associated genes
  • the present invention describes two members of this family, CTAGE-1 and CTAGE-2, their underlying cDNA
  • Cutaneous T-cell lymphoma is a tumor of the skin originating from T-lymphocytes that is difficult to treat in the advanced stage. Cutaneous T-cell lymphomas generally belong to the group of non-Hodgkin lymphomas. Cutaneous T-cell lymphomas include Mycosis fungoides diseases, Sezary syndrome and pagetoid reticulosis.
  • Conventional therapies radiation, chemotherapy
  • immunotherapy vaccination against tumors
  • the use of such forms of therapy requires tumor-specific antigens that have not been found for many types of tumor.
  • the invention is essentially based on the technical problem, including to find suitable tumor-specific antigens for cutaneous T-cell lymphomas that can be used in the context of vaccination therapy.
  • CTAGE-1 and CTAGE-2 are of interest because the proteins encoded and peptides derived therefrom are targeted, e.g. for cytotoxic cells, and can be used as antigens for the production of diagnostic or therapeutic antibodies.
  • the peptides or fragments thereof encoded by CTAGE-1 or CTAGE-2 can either be applied directly or loaded onto antigen-presenting cells.
  • the peptides representing antigens can also be expressed with the aid of vectors in different cells (e.g. dendritic cells as antigen-presenting cells).
  • the cloning of one or more representatives of the CTAGE gene family forms the basis for the development of diagnostic tests in order to ensure a more reliable and early diagnosis of those affected in the future.
  • functional analyzes of the protein will undoubtedly contribute to understanding tumor development. They are therefore to be regarded as candidate genes for studies of the pathomechanisms that underlie various tumor diseases.
  • the present invention thus relates to a DNA molecule coding for a tumor-associated antigen, which comprises:
  • nucleic acid molecules defined under (a) and (c) code for proteins, Polypeptides or peptides which have at least one of the biological activities described below of the protein encoded by the nucleic acid according to FIG. 1 or 2, for example a tumor-specific antigen.
  • the DNA molecules defined under (a) also include DNA molecules which are different from the sequence given in FIGS. 1 and 2 by deletion (s), insertion (s), exchange (s) and / or others in the state modifications known in the art, e.g. alternative splicing, distinguish or comprise a fragment of the original nucleic acid molecule, the protein encoded by these DNA molecules also having one or more of the biological activities described above. This also includes allele variants.
  • Methods for generating the above changes in the nucleic acid sequence are known to the person skilled in the art and are described in standard works in molecular biology, for example in Sambrook et al., Molecular Cloning: A Laboratory Manual, 2.
  • the variants have at least 70%, preferably 80%, very preferably 90, more preferably 95, 96, 97, 98 or 99% homology to the sequences according to FIG. 1 or 2.
  • hybridizing DNA refers to a DNA that hybridizes with a DNA of (a) under customary conditions, in particular at 20 ° C. below the melting point of the DNA.
  • hybridize refers to conventional hybridization conditions, preferably to hybridization conditions in which 5xSSPE, 1% SDS, IxDenhardts solution is used and the hybridization temperatures between 35 ° C and 70 ° C, preferably at 65 ° C , lie.
  • the DNA molecule according to the invention is a cDNA.
  • the DNA molecule according to the invention is a genomic DNA which is preferably derived from a mammal, for example a human. Screening methods based on nucleic acid hybridization allow the isolation of the genomic DNA molecules according to the invention from any organism or derived genomic DNA banks, using probes which contain the nucleic acid sequence shown in FIG. 1 or 2 or a part thereof.
  • a nucleic acid according to the invention is particularly suitable as an antigen-coding structure for therapeutic purposes.
  • the aim is to stimulate the immune system, to eliminate tumor cells that are identified by a member of the CTAGE family, in particular CTAGE-1 or CTAGE-2.
  • CTAGE-1 or CTAGE-2 There are different ways, e.g. Injecting the naked DNA into the patient.
  • a plasmid with a very active promoter and a member of the CTAGE family, in particular CTAGE-1 or CTAGE-2 is injected, for example, into the muscle or intradermally.
  • the aim is that the cells take up the plasmid, produce antigens, present individual peptides via HLA molecules and thus elicit a cytotoxic T-cell immune response. This should then lead to the defense against tumor cells.
  • the nucleic acids according to the invention can also be inserted into a vector or expression vector.
  • the present invention also encompasses vectors containing these nucleic acid molecules. Examples of such are known to the person skilled in the art.
  • vectors containing these nucleic acid molecules are known to the person skilled in the art.
  • these are, for example, pGEMEX, pUC derivatives (for example pUC8), pBR322, pBlueScript, pGEX-2T, pET3b and pQE-8.
  • pY100 and Ycpadl should be mentioned, for example, rend for expression in animal cells, for example pKCR, pEFBOS, cDM8 and pCEV4.
  • the baculovirus expression vector pAcSGHisNT-A is particularly suitable for expression in insect cells.
  • the nucleic acid molecule according to the invention is functionally linked in the vector to regulatory elements which allow its expression in prokaryotic or eukaryotic host cells.
  • regulatory elements for example a promoter
  • such vectors typically contain an origin of replication and specific genes which allow the phenotypic selection of a transformed host cell.
  • the regulatory elements for expression in prokaryotes for example E.
  • coli include the lac, trp promoter or T7 promoter, and for expression in eukaryotes the AOX1 or GAL1 promoter in yeast, and the CMV, SV40 , RVS-40 promoter, CMV or SV40 enhancer for expression in animal cells.
  • suitable promoters are the metallothionein I and the polyhedrin promoter.
  • Suitable vectors include in particular T7-based expression vectors for expression in bacteria (Rosenberg et al., Gene 56 (1987), 125) or pMSXND for expression in mammalian cells (Lee and Nathans, J.Biol.Chem. 263 (1988) , 3521).
  • the DNA according to the invention can be inserted into a vector not only for the purposes of recombinant production, but also in order to inject the DNA into patients with the aid of vectors, where it encodes an antigen for therapeutic purposes.
  • the DNA is antigen-presenting cells (APCs), e.g. Dendritic cells, for the HLA presentation of APCs.
  • the vector containing the DNA according to the invention can be injected in various ways: a) Lipid or liposome-packed DNA or RNA, for example generally described by Nabel et al., Proc. Natl. Acad. Be. USA, Vol. 93, pp. 15388-15393 (1996) b) With a bacterium as a transport vehicle for the expression vector. Suitable bacteria are, for example, (attested) listeria [eg Listeria monocytogenes], Salmonella strains [eg Salmonella spp.]. In general, this technique is described by Medina et al., Eur. J. Immunol., 29, pp. 693-699 (1999) and Guzman et al., Eur.
  • the vector containing the DNA molecules according to the invention is a virus, for example an adenovirus, vaccinia virus or an AAV virus, which is useful in gene therapy.
  • Retroviruses are particularly preferred. Examples of suitable retroviruses are MoMuLV, HaMuSV, MuMTV, RSV or GaLV.
  • the aforementioned viruses and fowlpox virus, canarypox virus, influenza virus or Sindbis virus are also suitable as the basis of a vaccine.
  • Such new vaccines which after administration to the patient anti-tumor
  • the DNA molecules according to the invention can also be transported to the target cells in the form of colloidal dispersions. These include, for example, liposomes or lipoplexes
  • vectors or plasmids which contain the DNA molecules according to the invention and suitable control sequences. These methods include, for example, in vitro recombination techniques, synthetic methods and in vivo recombination methods, as are described, for example, in Sambrook et al., Supra.
  • the present invention also relates to host cells containing the vectors described above.
  • host cells include bacteria, yeast, insect and animal cells, preferably mammalian cells.
  • the E. coli strains are preferred HB101, DH1, X1776, JM101, JM109, BL21, XLIBIue and SG 13009, the yeast strain Saccharomyces cerevisiae and the animal cells L, 3T3, FM3A, CHO, COS, Vero, HeLa and the insect cells sf9.
  • Methods for transforming these host cells, for phenotypically selecting transformants and for expressing the DNA molecules of the invention using the vectors described above are known in the art.
  • a plasmid is in vitro in an antigen presenting cell (APCs), e.g. Dendritic cells are given, which then produce antigens and present individual peptides via HLA molecules.
  • APCs antigen presenting cell
  • the plasmid DNA can be brought into the antigen-presenting cells in various ways: (a) as naked DNA, e.g. using "gene gun” or electroporation
  • the present invention also relates to a method for producing a protein which is encoded by the above nucleic acids, comprising culturing the above-described host cells under conditions which allow expression of the Allow protein (preferably stable expression), and recovery of the protein from the culture.
  • Suitable methods for the recombinant production of the protein are generally known (see, for example, Holmgren, Annu.Rev.Biochem. 54 (1985), 237; LaVallie et al., Bio / Technology 11 (1993), 187; Wong, Curr.Opin.Biotec - h.6 (1995), 517; Romanos, Curr.Opin.Biotech. 6 (1995), 527; Williams et al., Curr.
  • Suitable purification methods for example preparative chromatography, affinity chromatography, for example immunoaffinity chromatography, HPLC etc. are also generally known.
  • the present invention relates to a protein encoded by the DNA molecules according to the invention (CTAGE-1 or CTAGE-2) or obtained by the above method.
  • CTAGE-1 or CTAGE-2 The protein coding for CTAGE-2 is shown in FIG. 3.
  • the protein according to the invention is prepared according to conventional methods known in the art.
  • the exchanges preferably include "conservative" exchanges of amino acid residues, i.e. Exchanges for biologically similar residues, e.g. the substitution of a hydrophobic residue (e.g. isoleucine, valine, leucine, methionine) for another hydrophobic residue, or the substitution of a polar residue for another polar residue (e.g. arginine for lysine, glutamic acid for aspartic acid etc.).
  • Deletions can lead to the generation of molecules that are significantly smaller in size, i.e. those that have amino acids, for example
  • the variants have at least 70%, preferably 80%, very preferably 90, further preferably 95, 96, 97, 98 or 99% homology to the amino acid sequence which is derived from the nucleotide sequence according to FIG. 1 or to the amino acid sequence according to FIG. third
  • Injections of the protein or one or more peptides derived therefrom are also suitable for the desired anti-tumor vaccination. From the sequence of the protein according to the invention are determined either by means of appropriate computer programs or by means of experiments (eg phagocytotic uptake of the total protein, then analysis of the presenting peptides) HLA-dependent peptide fragments. These are artificially produced using methods known to the person skilled in the art and then (if necessary stimulating with an immune system
  • the protein according to the invention or fragments thereof can also be loaded onto APCs in vitro.
  • the loaded cells are then given to the patient e.g. injected into the lymph nodes and directly stimulate and proliferate tumor-specific cytotoxic T cells (Nestle et al., Nature Medicine, Vol. 4, No. 3, pp. 328 ff. (1998);
  • Whitney et al. in: Burg, Dummer, Strategies for Immunointerventions in Dermatology, Springer Verlag, Berlin Heidelberg, pp. 399-409, 1997)
  • the present invention also relates to antibodies which specifically recognize the protein CTAGE-1 or CTAGE-2 described above.
  • the antibodies can be monoclonal, polyclonal or synthetic antibodies or fragments thereof, for example Fab, Fv or scFv fragments. These are preferably monoclonal antibodies.
  • For the production it is favorable to immunize animals, in particular rabbits or chickens for a polyclonal and mice for a monoclonal antibody, with an above (fusion) protein or fragments thereof. Further "boosters" of the animals can be carried out with the same (fusion) protein or fragments thereof.
  • the polyclonal antibody can then be obtained from the serum or egg yolk of the animals.
  • the antibodies according to the invention can be produced according to standard methods, the protein encoded by the DNA molecules according to the invention or a synthetic fragment thereof serving as an immunogen.
  • Monoclonal antibodies can be produced, for example, by the method described by Köhler and Milstein (Nature 256 (1975), 495) and Galfre (Meth. Enzymol. 73 (1981), 3), in which mouse myeloma cells are fused with spleen cells derived from immunized mammals , These antibodies can be used, for example, for immunoprecipitation of the proteins discussed above or for the isolation of related proteins from cDNA expression banks.
  • the antibodies can be bound, for example, in liquid phase immunoassays or to a solid support.
  • the antibodies can be labeled in different ways.
  • Suitable markers and labeling methods are known in the art. Examples of immunoassays are ELISA and RIA.
  • the antibodies can also be used therapeutically.
  • a protein from the CTAGE family for example CTAGE-1 or CTAGE-2, serves as a target for bispecific antibodies.
  • CTAGE-1 or CTAGE-2 serves as a target for bispecific antibodies.
  • the administration of the antibodies can promote the tumor growth and metastasis-promoting effects of the CTAGE
  • CTAGE-1 or CTAGE-2 antisense DNA can inhibit the translation of CTAGE-1 or CTAGE-2 and thus have a therapeutic effect specifically on this gene.
  • RNA / DNA hybrids form in the corresponding tumor cells, which thus prevent transcription and at the same time bring about a breakdown of the hybrids (and thus the RNA) by RNase H (Scanion et al., The Faseb Journal, Vol. 9, p. 1288-1296, 1995)
  • the present invention further relates to the use of the DNA molecules, vectors, proteins and / or antibodies described above. Preferably these are used in the manufacture of a medicament for the diagnosis or treatment of tumor diseases in which CTAGE-1 or CTAGE-2 plays a role. It is preferred to provide a vaccination agent based on either the DNA or the protein / peptide as described above.
  • Suitable carriers and the formulation of such medicaments are known to the person skilled in the art. Suitable carriers include, for example, phosphate-buffered saline solutions, water, emulsions, for example oil / water emulsions, wetting agents, sterile solutions, etc.
  • Suitable carriers include, for example, phosphate-buffered saline solutions, water, emulsions, for example oil / water emulsions, wetting agents, sterile solutions, etc.
  • Drugs can be taken orally or parenterally.
  • Methods for parenteral administration include topical, intra-arterial, intramuscular, subcutaneous, intramedullary, intrathecal, intraventricular, intravenous, intraperitoneal, or intranasal administration.
  • the appropriate dosage is determined by the treating doctor and depends on various factors, for example the age, gender, weight of the patient, the stage of the disease, the type of administration, etc.
  • the present invention further relates to a diagnostic composition containing the DNA molecule or antibody described above, or
  • the diagnostic composition is suitable on the one hand to determine a tumor disease, but also to carry out a follow-up.
  • the DNA molecule according to the invention can also be used as a probe to isolate DNA molecules which originate, for example, from another species or another organism and encode a protein with the same biological activity.
  • the probe preferably has a length of at least 10, particularly preferably at least 15 bases.
  • Suitable detection methods based on hybridization are known to the person skilled in the art, for example Southern or Northern blot.
  • Suitable markings for the Probes are also known to the person skilled in the art and include, for example, labeling with radioisotopes, bioluminescent, chemiluminescent, fluorescent labels, metal chelates, enzymes, etc.
  • the present invention relates to a method for the diagnosis of tumor diseases, in vitro, comprising the following steps: isolation of nucleic acid from the patient,
  • Size-separating gels for example agarose gels, the preparation and labeling of the probe and the detection of the hybridization, for example by means of "Southern blot" or in-situ hybridization.
  • the above detection can also be carried out using PCR or LCR.
  • Primers are used which flank the sequence according to the invention or suitable subregions. Diagnostically important are amplification products of DNA from the tissue in question which differ from the amplification products of DNA from healthy tissue with regard to the appearance of CTAGE-1 or CTAGE-2 specific bands.
  • RNA or poly (A) + RNA from biological samples
  • separation of the RNAs on size-separating gels for example denaturing agarose gels
  • production and labeling of the probe and the detection be applied via "Northern blot”.
  • a possible disease can also be diagnosed by a method which comprises the following steps:
  • CTAGE-1 or CTAGE-2 indicates what is indicative of a tumor disease.
  • This detection can also be performed using standard techniques known to those skilled in the art. These are also known cell disruption methods which allow the isolation of the protein in such a way that it can be brought into contact with the antibody.
  • the detection of the bound antibody is preferably carried out using immunassays, for example
  • the antibodies are furthermore suitable for intercepting CTAGE-1 or CTAGE-2 overexpressed in tumors and thus inhibiting tumor growth, since there are indications that the presence of CTAGE-1 or CTAGE-2 is not only indicative of the presence of tumors indicates, but actively promotes tumor growth.
  • kits for carrying out the diagnostic methods according to the invention which contain the antibody according to the invention or a fragment thereof, a DNA molecule according to the invention as a probe or for
  • PCR or LCR suitable primer pair based on the sequence of the DNA molecule according to the invention, optionally in combination with a suitable detection means.
  • the DNA molecules, antibodies or fragments thereof contained in the kit can be immobilized on a suitable carrier.
  • a cDNA phage bank (in Lambda ZAP) was produced from mRNA of a healthy testis (Uni-ZAP TM XR Custom cDNA Library, Stratagene, Cat # 837201).
  • the bank was searched using the so-called SEREX method (Sahin et al., Proc. Natl.
  • Lymphomas mycosis fungoides, Sezary syndrome
  • the DNA of FIG. 1 is provided with BamHI linkers, cut with BamHI and inserted into the expression vector pQE-8 (Qiagen) cleaved with BamHI.
  • the expression plasmid pQ / CTAGE-1 is obtained.
  • pQ / CTAGE-1 is used to transform E.coli SG 13009 (see. Gottesmann, S. et al., J. Bacteriol. 148, (1981), 265-273).
  • the bacteria are cultivated in an LB medium with 10 ⁇ g / ml ampicillin and 25 ⁇ g / ml kanamycin and induced for 4 h with 60 ⁇ M isopropyl- ⁇ -D-thiogalactopyranoside (IPTG).
  • IPTG isopropyl- ⁇ -D-thiogalactopyranoside
  • chromatography Ni-NTA resin
  • the bound fusion protein is eluted in a pH 3.5 buffer. After neutralization, the fusion protein is subjected to 18% SDS polyacrylamide gel electrophoresis and stained with Coomassie blue
  • a fusion protein according to the invention from Example 2 is subjected to an 18% SDS polyacrylamide gel electrophoresis. After staining the gel with 4 M sodium acetate, the corresponding band is cut out of the gel and incubated in phosphate-buffered saline. Pieces of gel are sedimented before the protein concentration of the supernatant is determined by SDS-polyacrylamide gel electrophoresis followed by a Coomassie blue stain.
  • the rabbit's serum is tested in an immunoblot.
  • a fusion protein according to the invention from Example 2 is subjected to SDS-polyacrylamide gel electrophoresis and transferred to a nitrocellulose filter (cf. Khyse-Andersen), J., J. Biochem.Biophys. Meth. 10, (1984), 203-209).
  • the nitrocellulose filter is incubated with a first antibody at 37 ° C. for 1 h.
  • This antibody is rabbit serum (1: 10000 in PBS).
  • the nitrocellulose filter is incubated with a second antibody.
  • This antibody is a monoclonal goat anti-rabbit IgG antibody (Dianova) (1: 5000) coupled with alkaline phosphatase in PBS.
  • Antibodies are extracted from egg yolk and tested in a Western blot. Polyclonal antibodies according to the invention are detected.
  • Northern blots with mRNAs from various healthy tissues were carried out at 50 ° C. overnight in a hybridization buffer with 2xSSC, 10-fold Denhardt's solution, 10% dextran sulfate, 1% SDS, 3% SSC and 0.1 mg / ml salmon sperm DNA and the specific probe hybridized.
  • the specific probe was prepared by radioactive labeling (random-primed DNA labeling kit; Roche Diagnostics) of the PCR product from a PCR on Testis control cDNA, as described below under Example 5. The filters were washed once at 65 ° C in 2xSSC / - 0.1% SDS and in 0.2 x SSC / 0.1% SDS and with them at -70 ° C
  • AMV reverse transcriptase with the addition of 1.6 ⁇ g RNAse inhibitor was used in a total volume of 20 ⁇ l. The procedure followed the manufacturer's instructions.
  • the cDNA obtained from the RNA was checked for the presence of CTAGE-1 using the polymerase chain reaction using a CTAGE-1 specific primer.
  • the PCR conditions were:
  • Primer 1 5'-CTC CTG ACT TCT TTC CCA ACC TTT ACC-3 '
  • Primer 2 5'-TGC AAT TCC CAC CTA ACT TCC ATT CTG-3
  • Intestine small intestine, fetal liver, fetal lungs, fetal spleen, fetal kidney, fetal skeletal muscle, fetal thymus, fetal brain, fetal heart, brain, skin, heart, bone, liver, lungs, stomach, spleen, kidney, ovary, pancreas , Peripheral blood lymphocytes, placenta, prostate, skeletal muscle, T-lymphocytes (activated), thymus, trachea.
  • Mycosis fungoides 4 of 13 positive T-zone lymphoma: 1 of 1 positive Sezary syndrome: 1 of 2 positive CD3O lymphoma; 0 out of 1 positive
  • CTAGE-2 was identified by means of Race PCR (Clontech, Heidelberg).
  • the primers should be approx. 23-28 nucleotides long and should be chosen with a very high annealing temperature (ideally above 70 ° C).
  • the primer extending in the 5 'direction should be near the 5' end, the 3 'extending near the 3'
  • the choice of primer lies in the knowledge of the expert.
  • the 5 'extending primer is a reverse primer and is called Race 1
  • the 3' extending primer is a forward primer and is called Race 2.
  • nested race modified pipetting scheme: instead of 5 ⁇ l UPM, 1 ⁇ l NUP (nested universal primer mix) instead of race 1, nested race ß modified PCR program: instead of touch down PCR, 15 cycles nes-Race
  • 3'cDNA synthesis uses a normal reverse transcriptase reaction with an initial oligo dT primer that hybridizes at the poly A end.
  • the 3 'CDS (dT OligoMix) mix also contains the Smart Oligoll (in lower concentration) (see 5.2.2), so that a 5' full length polymerization takes place.
  • 5'cDNA synthesis like the 3 'synthesis, is started by an oligo dT (5'CDS mix) at the 3' end of the mRNA.
  • the Smart II Oligo system takes advantage of the special feature that the reverse transcriptase MMLV, after a full length transcription, is a terminal transferase
  • the SMART II oligo can bind to this cytosine-rich region because it has a guanine-rich region.
  • a known sequence Smart II Oligo
  • the SMART II Oligo now serves as a primer for reverse transcription in the 3 'direction.
  • the MMLV transcriptase only attaches to such a cytosine-rich region to polymerized mRNAs, so that this ensures that only full-length cDNA are formed.
  • the antibody causes an automatic hot start PCR.
  • the monoclonal antibody binds to the N terminus of the Taq polymerase and thus inhibits its activity.
  • the antibody When heated to 95 ° C initially, the antibody is denatured and the polymerase is no longer inhibited.
  • the race PCR takes advantage of the fact that the SMART fill length cDNA is limited by known sequences. These serve as forward / reverse primers for the amplification
  • the known sequence attached to the oligo dT in the 3 'CDS primer mix is the same as that which is the SMART Oligo II tag attached to the guanine rich sequence.

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Abstract

L'invention concerne une nouvelle famille génique pour </= gènes associés à l'érythrodermie de type Sézary >/= (=cutaneous T-cell lymphoma associated genes) (CTAGE). L'invention concerne deux membres de cette famille : CTAGE-1 et CTAGE-2, leurs gènes sous-jacents et leur utilisation à des fins de diagnostic et de traitement d'affections tumorales.
PCT/DE2000/003628 1999-10-14 2000-10-13 Famille genique pour ctage Ceased WO2001027255A2 (fr)

Priority Applications (4)

Application Number Priority Date Filing Date Title
DE50014417T DE50014417D1 (de) 1999-10-14 2000-10-13 Nukleinsäure, die für ein protein der ctage-familie kodiert, deren verwendung und herstellung
EP00983011A EP1230265B1 (fr) 1999-10-14 2000-10-13 Famille genique pour ctage
AU19936/01A AU1993601A (en) 1999-10-14 2000-10-13 Ctage gene family
US10/110,807 US7314922B1 (en) 1999-10-14 2000-10-13 Ctage gene family

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DE19949595A DE19949595A1 (de) 1999-10-14 1999-10-14 CTAGE-Genfamilie
DE19949595.5 1999-10-14

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WO2001027255A2 true WO2001027255A2 (fr) 2001-04-19
WO2001027255A3 WO2001027255A3 (fr) 2001-11-22

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DE10055285A1 (de) * 2000-11-08 2002-06-06 Deutsches Krebsforsch Neue Marker für die Diagnose und Therapie von Tumoren

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US5804381A (en) * 1996-10-03 1998-09-08 Cornell Research Foundation Isolated nucleic acid molecule encoding an esophageal cancer associated antigen, the antigen itself, and uses thereof
US5830702A (en) 1990-10-31 1998-11-03 The Trustees Of The University Of Pennsylvania Live, recombinant listeria monocytogenes and production of cytotoxic T-cell response
US6051237A (en) * 1994-11-08 2000-04-18 The Trustees Of The University Of Pennsylvania Specific immunotherapy of cancer using a live recombinant bacterial vaccine vector
US6617156B1 (en) * 1997-08-15 2003-09-09 Lynn A. Doucette-Stamm Nucleic acid and amino acid sequences relating to Enterococcus faecalis for diagnostics and therapeutics
US6297364B1 (en) * 1998-04-17 2001-10-02 Ludwig Institute For Cancer Research Isolated nucleic acid molecule encoding cancer associated antigen, the antigen itself, and uses thereof
US6812339B1 (en) * 2000-09-08 2004-11-02 Applera Corporation Polymorphisms in known genes associated with human disease, methods of detection and uses thereof

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE10055285A1 (de) * 2000-11-08 2002-06-06 Deutsches Krebsforsch Neue Marker für die Diagnose und Therapie von Tumoren

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DE19949595A1 (de) 2001-05-03
AU1993601A (en) 2001-04-23
WO2001027255A3 (fr) 2001-11-22
EP1230265A2 (fr) 2002-08-14
EP1230265B1 (fr) 2007-06-13
ATE364626T1 (de) 2007-07-15
US7314922B1 (en) 2008-01-01
DE50014417D1 (de) 2007-07-26
ES2288884T3 (es) 2008-02-01

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